Method for promoting hard tissue formation

ABSTRACT

Formulations and methods to promote biological processes to form or regenerate new hard tissues such as bones, cartilage, and/or dental tissues are disclosed. The formulation comprising two proteins is administered to enhance biological activities of a hard tissue growth and differentiation factor characterized by specific and selective upregulation and/or extension of the retention time of the intracellular enzymes and signaling molecules that play important roles to proliferate, differentiate, maturate, and/or mineralize the hard tissue forming cells.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application Nos.60/805,201 filed Jun. 19, 2006, 60/803,327 filed May 26, 2006,60/747,255 filed May 15, 2006, 60/747,143 filed May 12, 2006 and60/700,518 filed Jul. 18, 2005 all of which applications areincorporated herein by reference in their entirety noting the presentapplication controls in the event of any conflict which an earlierapplication.

FIELD OF THE INVENTION

The present invention relates generally to formulations and methods fortreating hard tissue by administering a combination of two or morepeptides.

BACKGROUND OF THE INVENTION

It is well-documented that disorders of bone tissues causes numeroussignificant health problems on a world-wide basis. Because of suchsignificant health problems with bone diseases, numerous efforts havebeen made to develop new therapeutic agents for bone disorders.

For example, several growth and/or differentiation factors are known toeffect bone, cartilage, and dental tissues. Many of these have beenevaluated for their ability to speed or alter the healing of defects inthese tissues. Such factors include the molecules belonging to thetransforming growth factor (TGF) and bone morphogenetic protein (BMP)family as well the epideimal growth factor (EGF), epithelial cell growthfactor (ECGF), fibroblast growth factor (FGF), platelet derived growthfactor (PDGF), insulin-like growth factor (IGF), and insulin-like growthfactor binding protein (IGFBP) families.

Although many of these factors are known to promote proliferation,differentiation, maturation, or mineralization of osteoblastic cells,attempts to develop these factors as novel therapeutics have beenlimited by their lack of tissue specificity. Administration of thesefactors affect tissues other than skeletal tissues, which can result inundesirable activities.

For example, the administering of rhBMP-2 to soft tissue (e.g.subcutaneous injection) causes rapid formation of new bone in softtissue. As a result of this response, use of rhBMP-2 has been limitedand needs to be carefully applied in order to prevent calcification inundesirable locations.

A further limitation of using the above growth and/or differentiationfactors as a therapeutic is the cost of manufacturing. The factors areall proteins manufactured by recombinant DNA methods, which requirelarge-scale fermentation or cell culture processes. In addition, thesemanufacturing methods require highly specialized facilities, whichfurther increase the cost of manufacturing. As a result, themanufacturing cost gets translated into a very expensive cost oftreatment for using these products.

For example, a locally implantable collagen sponge that contains a BMPfamily molecule has been used as a medical device for spinal fusiontherapies. However, the cost for such a procedure has provided alimitation of its availability to patients who could benefit from thistype of treatment.

Accordingly, there is continued interest in the development of newtherapeutics that would significantly reduce the cost of treatment. Ofparticular interest would be a novel therapeutic that would reduce therequired dose of the already existing therapeutics.

Furthermore, growth factors other than those in the BMP family, such asthose in the TGF, PDGF, EGF, FGF, and IGF families, should be exploredas potential therapeutics. The utilization of other growth factors thatreduce the efficacious dose and cost of therapies for existing growthfactors therapeutics would be of great value to the orthopedic andrelated medical community and the patients that they serve.

SUMMARY OF THE INVENTION

The present invention relates to the formation or regeneration of newhard tissues such as bones, cartilage, and/or dental tissues. Theinvention comprises administering a first peptide which enhances theactivity of a second peptide. More specifically, the first peptideenhances the biological activities and/or therapeutic effects of asecond peptide which may be a hard tissue growth factor and/ordifferentiation factor. The first peptide and the second peptide may betogether within a single formulation. Alternatively, the first andsecond peptides may be administered at substantially the same time orsequentially in either order. The first and second peptides may bepresent in the formulation or administered in substantially the sameamount or different ratios relative to each other.

The first peptide comprises 10 to 50 amino acids having the amino acidsequence RGDBDXnSGZG, and wherein B, X, and Z are chosen from any aminoacid residue and n is an integer between 1 and 10 (SEQ ID NO:3). Thefirst peptide (e.g. a peptide of SEQ ID NO:8) is administered in anamount so as to enhance a characteristic of a second peptide chosen fromdifferentiation, proliferation, maturation and mineralization of cellsinvolved in the formation of hard tissue.

The second peptide (e.g., rhBMP-2) may be a growth factor which belongsto the family of transforming growth factor-beta (TGF-β). The TGF-β maybelong to a family of bone morphogenic proteins (BMP).

Specific examples of the first peptide and the second peptide areprovided here. The two peptides may be administered in a singleformulation and the formulation may include the first and secondpeptides in equal amounts or different ratios. More specifically,formulations of the invention may be comprised of a pharmaceuticallyacceptable carrier (e.g. an absorbable collagen sponge (ACS)) and twoproteins wherein the first protein is a protein chosen from

RGDBD(X)nSGZG (SEQ ID NO.1) [B, X, and Z can be any amino acid, n= 1~10]] RGDND(X)nSGZG [X and Z can be any amino acid, n = (SEQ ID NO.2)1~10] RGDND(X)nSGDG [X can be any amino acid, n = 1~10] (SEQ ID NO.3)RGDNDJJPFSGDG (SEQ ID NO:4) [J can be any amino acid] RGDNDISPFSGDG (SEQID NO.5) RGDNDMSPFSGDG (SEQ ID NO.6) RGDNDVPPFSGDG (SEQ ID NO.7)TDLQERGDNDISPFSGDGQPFKD (SEQ ID NO.8)and the second protein is chosen from molecules belonging to thetransforming growth factor (TGF) and bone morphogenetic protein (BMP)family (e.g. rhBMP-2) as well the epidermal growth factor (EGF),epithelial cell growth factor (ECGF), fibroblast growth factor (FGF),platelet derived growth factor (PDGF), insulin-like growth factor (IGF),and insulin-like growth factor binding protein (IGFBP) families.

An aspect of the invention is that peptides such as rhBMP-2 are quiteexpensive and by combining such a peptide with a first peptide of theinvention (e.g. a peptide of SEQ ID NO:8) it is possible to reduce theamount of the rhBMP-2 which is administered while obtainingsubstantially the same the desired therapeutic results as compared toadministering a larger amount of rhBMP-2 without the first peptide. Thesecond peptide may be the commercial available recombinant human bonemorphogenic protein-2 (rhBMP-2) which may be on a commercially availabletype I bovine absorbable collagen sponge (ACS) sold as Helistat® byIntegra Life Sciences, Plainsboro, N.J. The first peptide of theformulation which may be a peptide of SEQ ID NO:8 can be manufacturedinexpensively in relatively large amounts. Thus, although the first andsecond peptides could be administered in equal amounts or differentamounts there are economic reasons for administering the first peptideof the formulation in a relatively large amount relative to the secondpeptide. Peptides such as rhBMP-2 are very expensive to produce relativeto the cost of producing a peptide of SEQ ID NO:8 Accordingly, if thefirst peptide is a peptide such as that of SEQ ID NO:8 and the secondpeptide is a peptide such as a bone morphogenic protein (rhBMP-2) theratio of the second peptide to the first peptide may be any ratio whichobtains an improved result compared to the same amount of only one ofthe peptides being used. However, because the second peptide such asrhBMP-2 is substantially more expensive as compared to the peptide ofSEQ ID NO:8 there are economic reasons for using the two peptides in aformulation such that the first peptide such as the peptide of SEQ IDNO:8 is present in a larger amount relative to the second peptide suchas rhBMP-2. Assuming the first number in the ratio represents the firstpeptide such as peptide of SEQ ID NO:8 and the second number representsan amount of the second peptide such as rhBMP-2, the formulation may becreated where the ratio of the first peptide to the second peptide is1:1 or more and that ratio can be extended up to 1:5,000 and possiblymore. Ratios in intermediate amounts can also be used such as 1:2through 1:5,000. Other ratios such as 1:5, 1:10, 1:50, 1:200, 1:300,1:500, could be used and those skilled in the art could readilydetermine within a particular situation and formulation the desiredratio in terms of obtaining the most therapeutic effect at the mosteconomic cost. The ratios of one protein or peptide to another areratios based on the weight of the protein or peptide used in theformulation.

A specific example of the invention is a formulation which isspecifically designed for promoting hard tissue formation andregeneration. The formulation may be comprised of a pharmaceuticallyacceptable carrier such as an injectable carrier having therein thefirst protein and the second protein of the invention. Still morespecifically, the formulation may be comprised of a pharmaceuticallyacceptable carrier which may by an ACS or an injectable carrier a firstprotein having the SEQ ID NO:8 and the second protein in the form ofrhBMP-2. The first and second proteins may be present in equal amountsor in any different ratios including those indicated above. Further,although specific formulations described here include a single firstprotein and a single second protein it is understood that multipleproteins from each group may be included within a formulation or may beadministered separately, at the same time or sequentially at differenttimes.

An aspect of the invention is that by including the first peptide of theinvention it is possible to administer the second peptide in an amountwhich would be below levels which would (by itself) be expected toprovide a therapeutic result. Thus, a formulation can be prepared bycombining a pharmaceutically acceptable injectable carrier with apeptide of SEQ ID NO:8 and rhBMP-2 wherein the amount of rhBMP-2 is anamount which would not be therapeutically effective in the absence ofthe peptide of SEQ ID NO:8.

A method to promote biological processes to form or regenerate new hardtissues such as bones, cartilage, and/or dental tissues is disclosedwherein the method may be carried out at substantially reduced costs ascompared to the use of rhBMP-2 alone. More specifically, the presentinvention is a method of enhancing the biological activities of a hardtissue growth and differentiation factor characterized by specific andselective upregulation and/or extension of the retention time of theintracellular enzymes and signaling molecules that play important rolesto proliferate, differentiate, maturate, and/or mineralize hard tissueforming cells. This invention provides the medical community with amethod of treating hard tissue disorders and of improving the efficacyof currently used practices. This method is also expected to result in asignificantly reduced cost to patients. By administering the firstprotein which may be a protein such as that of SEQ ID NO:8 it ispossible to reduce the amount of the second protein such as rhBMP-2 andstill obtain the desired therapeutic effect that would be obtained whenadministering optimal concentrations of rhBMP-2.

Another aspect of the invention is a kit for administration to thepatient. The kit may comprise instructions with respect to how tocombine and administer the components. The components may include apharmaceutically acceptable carrier. The carrier may be an injectablecarrier or it may be an implantable absorbable material such as animplantable absorbable sponge including the type I bovine absorbablecollagen sponge (ACS). Further, the kit may include a pre-measured andpackaged amount of a first peptide which may be any first peptide of thetype described herein including a first peptide of SEQ ID NO:8. The kitmay further include a second peptide which may be any second peptide ofthe type described herein which may include rhBMP-2. The kit may includemultiple different versions of the first peptide. Further, the kit mayinclude multiple different versions of the second peptide. In oneversion the kit includes separately packaged components of the carrierin the form of the absorbable collagen sponge, a first peptide of SEQ IDNO:8 and a second peptide which is rhBMP-2 wherein the peptide of SEQ IDNO:8 and rhBMP-2 are present in measured amounts in a ratio capable ofobtaining a desired therapeutic effect in an economically advantageousmanner.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the inventions as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

In accordance with the present invention, the invention is bestunderstood from the following detailed description when read inconjunction with the accompanying drawings. It is emphasized that,according to common practice, the various features of the drawings arenot to-scale. On the contrary, the dimensions of the various featuresare arbitrarily expanded or reduced for clarity. Included in thedrawings are the following figures:

FIG. 1 is a bar graph showing the dose dependent upregulation ofalkaline phosphatase (ALP) activity in mouse primary osteoblastscultured with a suboptimal concentration of rhBMP-2 when a differentconcentration of the peptide of SEQ ID No. 8 were added to the cultures.

FIG. 2 is a bar graph showing a dose dependent production ofprostaglandin E2 by MC3T3 cells cultured with a suboptimal concentrationof rhBMP-2 plus varying doses of the peptide of SEQ ID No. 8 was addedto the culture.

FIG. 3 is an image of a gel which demonstrates the expression ofphosphorylated Smads 1, 5, and 8 (as a total) indicated by Westein blotfrom MC3T3 cells at different time points. Cells were cultured with asuboptimal concentration of rhBMP-2 either with or without the peptideof SEQ ID No. 8.

FIG. 4 is a bar graph which production of PGE2 by calvaria when it wasisolated and cultured with different doses of the peptide of SEQ ID No.8.

FIG. 5 is a bar graph which exhibits various bone parameters such as newbone volume, cell density in the bone tissue, and osteoblast surface inthe mouse calvaria organ culture with the peptide of SEQ ID No. 8 ofrhBMP-2.

FIG. 6 shows bar graphs and images which represent radiographs(indicating new calcified tissue/bone) formed in the dorsal muscle ofmice when a polymer which containing rhBMP-2 and different doses of thepeptide of SEQ ID No. 8 were implanted in the tissue. (time point)

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods and formulations of the invention aredescribed, it is to be understood that this invention is not limited toparticular embodiments described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupercedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aprotein” includes a plurality of such proteins and reference to “thepeptide” includes reference to one or more peptides and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

The terms “treatment”, “treating” and the like are used here to describeobtaining a desired pharmacological and/or physiological effect whicheffect is an effect on hard tissue formation, regeneration, growth andthe like. The effects may be prophylactic in terms of completely orpartially preventing hard tissue loss and/or may be therapeutic in termsof partially or completely curing an adverse condition which isattributed to hard tissue loss. Accordingly, “treatment” as used here isintended to cover treating a mammal and in particular a human toinclude:

(a) preventing a disease or condition from proceeding in manner whichprevents further loss of hard tissue or further acceleration loss ofhard tissue;

(b) inhibiting a disease or condition, i.e. arresting a condition whichleads to hard tissue loss; and/or

(c) relieving a disease or condition thereby causing regression of thedisease or condition and thereby promoting hard tissue formation,regeneration and/or growth. By carrying out the invention it is possibleto enhance differentiation, proliferation, maturation and/ormineralization of cells involved in the formation of hard tissue.

The terms “synergistic”, “synergistic effect” and the like are usedherein to describe improved treatment effects obtained by combining theadministration of one or more proteins. Although a synergistic effect insome fields is meant an effect which is more than additive (e.g. 1+1=3),in many fields relating to bone disease an additive (1+1=2) or even lessthan additive (1+1=1.6) effect can be interpreted as synergistic. Forexample, one cannot treat any bone disease merely by adding together twoknown pharmaceuticals each of which are 50% effective in treatment toobtain a 100% effective treatment. Thus, in some situations adding twocomponents together can actually have a negative effect and obtainresults which are less desirable than when either drug is used byitself. Further, as used herein synergistic means that one of thecomponents (e.g. rhBMP-2) may be used in a lesser amount when combinedwith a second component (e.g. SEQ ID NO:8) and still obtain the samedesired effect as if the rhBMP-2 component where present in a largeramount. Thus, in connection with the invention by combining the firstprotein such as that of SEQ ID NO.:8 with the second protein such asrhBMP-2 the amount of rhBMP-2 needed to obtain desired therapeuticresults can be reduced. That reduction may be 10%, 20%, 50%, 75% or moreas compared with the dosing needed to obtain the desired result withoutthe administration of the first protein. A synergistic effect is alsoobtained with respect to the economic value of formulations of theinvention. Thus, when very large amounts of the first protein such as aprotein of SEQ ID NO:8 are used very small amounts of the second proteinsuch as rhBMP-2 are used. Because the rhBMP-2 is substantially moreexpensive to manufacture as compared to a protein of SEQ ID NO:8synergistic economic results are obtained even when substantially thesame therapeutic results are obtained. Thus, when large amounts of thefirst protein such as a protein of SEQ ID NO:8 are used the amount ofthe second protein may be one half or less, one tenth or less or evenone hundredth or less of the dosing that might be required to obtain adesired therapeutic effect in the absence of the first protein of SEQ IDNO:8.

The terms “simultaneous” and “sequential” in terms of administeringformulations of the invention are used herein to mean that the firstpeptide and the second peptide may be administered at exactly the sametime or one after another with consideration to normal medicalprocedures and results being obtained. Thus, simultaneous may mean thatthe two compounds are present within a single formulation or that theyare administered at the same point of treatment. “Sequentially” may meanthat they are administered sequentially one after another from the samesyringe or from different syringes. What is important is that the firstand second peptides be administered in such a manner that the firstpeptide and the second peptide can have an opportunity tophysiologically interact in terms of the desired treatment being sought.

Invention in General

Formulations of the invention are administered for enhancing hard tissueformation. The formulations may be administered by implanting them afterputting drug on an absorbable collagen sponge as a carrier oradministered by injection with the proteins in a pharmaceuticallyinjectable carrier. The formulation may include one, two or moreproteins. In one embodiment the formulation administered to a humanpatient includes a first protein which is a protein which is encompassedby the general sequence of SEQ ID NO: 1, in combination with the secondprotein which is chosen from molecules belonging to the transforminggrowth factor (TGF) and bone morphogenetic protein (BMP) family (whichincludes rhBMP-2), as well the epidermal growth factor (EGF), epithelialcell growth factor (ECGF), fibroblast growth factor (FGF), plateletderived growth factor (PDGF), insulin-like growth factor (IGF), andinsulin-like growth factor binding protein (IGFBP) families.

Formulations of the invention used in treating a mammal may include thecarrier and only one protein (e.g. SEQ ID NO:8) and may be present in akit wherein a second protein (e.g. rhBMP-2) is also present in apre-measured amount and packaged with a pharmaceutically acceptablecarrier which may be an absorbable collagen sponge (ACS) or aninjectable carrier. The two proteins may be packaged together forsimultaneous or sequential administration. Formulations or kits of theinvention may be designed in a way so as to obtain a desired therapeuticresult while minimizing the amount of the second protein present in thekit or formulation. Because the first protein such as the protein of SEQID NO:8 is relatively inexpensive to produce and the second protein(e.g. rhBMP-2) is quite expensive those skilled in the art willrecognize that formulating to include large amounts of the first proteinand small amounts of the second protein can provide desired therapeuticresults in a manner which is highly economical.

The second peptide used in treating a mammal may be the commercialavailable recombinant human bone morphogenic protein-2 (rhBMP-2) whichmay be on a commercially available type I bovine absorbable collagensponge (ACS) sold as Helistat® by Integra Life Sciences, Plainsboro,N.J.

The method of the invention enhances the activities of growth and/ordifferentiation factors, that are known to promote one or more ofproliferation, differentiation, maturation, and mineralization of thecells in the hard tissue forming cell lineage including but not limitedto osteoblasts, odontoblasts, ameloblasts, and cementoblasts, andthereby promote new hard tissue formation.

In a particular embodiment of this invention, the method of thisinvention specifically enhances the activities of differentiationfactors that usually promote one or more of differentiation, maturation,and/or mineralization of the hard tissue forming cells.

The method is characterized by using a peptide containing amino acidsequence that is generally defined by the SEQ ID No. 1. It wasdiscovered that, by adding the peptide to an in vitro or in vivoskeletal tissue forming system, the hard tissue formation that waspromoted by hard tissue growth and/or differentiation factors wassignificantly enhanced and that the necessary amount of such factors wassignificantly reduced. Further, this significant synergistic effectbetween the hard tissue growth and/or differentiation factors and thepeptides containing SEQ ID No. 1 is highly selective to the hardtissues. In other words, this unique enhancing effect by the peptidescontaining SEQ ID No. 1 is expected only in a tissue that has committedto become a hard tissue.

This unexpected and significant finding provides the medical communitywith an improved method to heal a hard tissue defects. The use of suchpeptides minimizes the necessary amount of such growth and/ordifferentiation factor required to exert maximum activity, therebyreducing the overall cost of treatment significantly.

Any of the peptides containing SEQ ID No. 1 can be manufactured bywidely used chemical synthesis methodology of the peptides and the costof goods thereof can be significantly lower than recombinant growthand/or differentiation factors.

In addition, because the biological activities of the peptidescontaining SEQ ID No. 1 are expressed only in a hard tissue environment,the administration route of the peptides to the subjects in need is notlimited even if the other growth and/or differentiation factors may havecertain limitation in their method of administration. In other words,the peptides containing SEQ ID No. 1 can be formulated and administeredin variety of manners depending upon the therapeutic procedure.

Another characteristic feature of this invention is that by adding thepeptides containing SEQ ID No. 1, the expression and activities ofcyclooxygenase-2 (COX-2) in the hard tissue forming cells such asosteoblasts, odontoblasts, cementoblasts, and ameloblasts, aresignificantly upregulated. This significant upregulation of COX-2 isobserved in a dose dependent manner following addition of the peptidecontaining SEQ ID No. 1.

Furthermore, the production of prostaglandin E2 (PGE2) by such hardtissue formation cells is significantly upregulated by the method.

It has been known that PGE2 promotes the proliferation of hard tissueformation cells. Therefore, the local upregulation of PGE2 in hardtissue formation cells is desirable.

Nagel, et. al. demonstrated that the peptide of SEQ ID No. 8, which isalso called AC-100 and under Phase II clinical developments for multiplehard tissue treatments, upregulated COX-2 in human mesenchymal stemcells (Journal of Cellular Biochemistry, 2004).

Also, Middleton-Hardie, et. al. has shown that the peptide of SEQ ID No.8 dose dependently upregulated the production of PGE2 in mouse calvariaorgan culture assay (27th Annual Meeting of the American Society forBone and Mineral Research 2005, in press). It has been also shown thatsome hard tissue growth factors such as BMP increased the production ofPGE2 by osteoblastic cells.

However, when the peptides containing the SEQ ID No. I are used with asuboptimal concentration of rhBMP-2, which marginally upregulated COX-2or PGE2 alone, either COX-2 or PGE2 were significantly upregulated in adose dependent manner of the peptides following the addition of thepeptides containing SEQ ID No. 1.

For example, addition of the peptide of SEQ ID No. 8 alone to humanmesenchymal cells in culture did not result in a significant increase inPGE2. Since enhancement of PGE2 occurs in organ cultures and whenrhBMP-2 is present (even at suboptimal concentrations), it is believedthat the peptide of SEQ ID No. 8 acted by substantially enhancing thedifferentiation activities of rhBMP-2 (whether added exogenously orproduced locally in bone or by bone cells).

Smad molecules (1˜8) are known to play important roles in intracellularsignaling of members of the TGF-β and BMP families. In hard tissueforming cells,

Smads 1, 5, and 8 are known to be activated by differentiation factorssuch as BMP family molecules. Such activation of Smad molecules can beobserved via a phosphorylation event that occurs within 10-20 minutesafter stimulation. Typically, the retention time of such phosphorylatedSmad molecules is short lived, usually disappearing within about 0.5 to12 hours after the initial stimulation of the cells.

Bone forming cells, such as the MC3T3 cells are stimulated with bothrhBMP-2 and a peptide containing SEQ ID No. 1, intracellular Smadmolecules (in particular Smads 1, 5, and 8) are phosphorylated andremain phosphorylated for up to 48 hours. This strongly suggests thatpeptides containing SEQ ID No. 1 have the ability to extend the activityof hard tissue forming cells and thereby enhance the hard tissueformation.

This correlation between the enhancement of hard tissue formation andextension of the retention time of the phosphorylated Smad molecules isextremely useful to design or identify a novel molecule that can enhancehard tissue formation. Those skilled in the art can establish an assaysystem to observe the retention time of the phosphorylated Smadmolecules in a hard tissue forming cell and use it to screen a newchemical entity that stimulates hard tissue formation.

Any compound that can extend the retention time of the phosphorylatedSmad molecule in hard tissue forming cells and a method to identify sucha compound using the screening system to evaluate the retention time ofthe phosphorylated Smad molecule in hard tissue forming cells are withinthe scope of this invention.

It has been known that BMP causes bone formation when administered orimplanted in mammals when given. The activity can be enhanced when givenin the appropriate delivery vehicle. As indicated in Example 6, new bonetissue was locally formed in about 20 days after a polymer comprisingpolylactic acid (PLA), polyethylene glycol (PGE), and a linker (DX)containing 5 μg or higher amount of rhBMP-2 was implanted to mousedorsal muscle. With lower, suboptimal doses of rhBMP-2, the there isminimal or no bone formation. Specifically in this case when a 3 μg dosewas used, there was substantially less bone formation compared toadministration of a 5 μg dose.

Varying amounts of peptide of SEQ ID No. 8 were added to the samepolymer containing as suboptimal dose, 3 μg of rhBMP-2. However, asindicated in FIG. 3, when a peptide of SEQ ID No. 8 dose was added tothe suboptimal dose of rhBMP-2, there was a significant and dosedependent increase in bone formation. When given at 50 μg/mL or higher,the degree of new bone formation was similar or superior to the amountof bone formed using an optimal dose of rhBMP-2.

Equivalent or comparable results are obtained in the same experimentswhen different peptides that have the amino acid sequence defined by SEQID No. 1 are used.

These in vivo results clearly suggest that a peptide containing SEQ IDNo.1 can significantly reduce the required amount of rhBMP-2 when it isused with such peptide to form a new bone in the mammals. Thissignificant reduction of the required amount of rhBMP-2 is extremelybeneficial to the medical practice due to a lower cost and a lower riskof ectopic calcification form higher doses of rhBMP-2.

Since hard tissue forming cells are in the same lineage, a similarsynergistic effect is expected in dental hard tissues such as dentin,enamel, and cementum, which are formed by odontoblasts, ameroblasts, andcementum, respectively.

The co-use of the peptides containing SEQ ID No. 1 for above hard tissueformation purposes is not limited to use with BMP family molecules. Asimilar effect can be expected when the peptides are used with otherhard tissue growth or differentiation factors including but no limitedto the molecules belonging to the families of TGF (BMP family is a partof it), EGF, PDGF, FGF, IGF, and IGFBPs.

The peptides used herein are defined by their containing of specificamino acid sequences specified by SEQ ID No. 1. The peptides can be inthe sizes of approximately 10 to 50 amino acids as far as they containthe amino acid sequence specified by SEQ ID No. 1. The peptides used inthis invention are not necessarily linear peptides but can be cyclic orbranched peptides. A peptide longer than 50 amino acids that contains atandem sequence of the sequence units of this invention is also withinthe scope of this invention. Multimers of the peptides containing SEQ IDNo. 1 are also within the scope. Such multimers can be dimers, trimers,tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers,etc.

The minimum size of the peptide of this invention has an amino acidsequence of RGDBDXSGZG wherein B, X, and Z can be any amino acid. Thepeptides containing the general amino acid sequence of SEQ ID No. 4demonstrate higher biological activities than other sequences for thepurpose of this invention whereas SEQ ID No. 8 exhibits the highestactivities.

The SEQ ID No. 5 and 8 are equivalent to the amino acid number 247-259and 242-264, respectively, of a known human protein named matrixextracellular phosphocglycoprotein (MEPE). The SEQ ID No. 6 is a macaquemonkey orthologue of the human sequence of SEQ ID No. 5. The SEQ ID No.7 is rodent (common between mouse and rat) orthologue of the sameportion. All of these orthologues of MEPE contain sequences within thescope of the SEQ ID No. 1 that promote proliferation, differentiation,maturation, and mineralization of hard tissue forming cells in anequivalent manner.

In summary, a method to promote or enhance new hard tissue formation waspresented in that a peptide comprising about 10 to 50 amino acid thatcontains amino acid sequence specified by SEQ ID No. 1. This peptidereduced the quantity of a bone differentiation and/or formation factor(e.g. rhBMP-2) needed to promote effective new bone formation. Furtherinvestigation suggests that this may be occurring by a mechanisminvolving extension of the life time of specifically phosphorylated Smadmolecules known to be involved in the molecular signaling pathwayresponsible for bone formation.

Kits

One kit in accordance with the invention includes one or more absorbablecollagen sponges of the type generally used commercially in connectionwith rhBMP-2. The sponges are present with a solution of the firstprotein such as a peptide of SEQ ID NO:8 and a separate containercomprising the second protein such as rhBMP-2. In another version of thekit the first protein such as the peptide of SEQ ID NO:8 and the secondprotein such as rhBMP-2 are present in the same solution container.Including the two proteins in different containers is advantageous inthat if the shelf life of the two containers is different it may not benecessary to discard both containers when the shelf life of only one hasexpired. Further, the kits can be designed so that the absorbablecollagen sponge or sponges are of the appropriate size to absorb theamount of solution from one or both of the containers containing thefirst and second peptides. Still further, the first and second peptideswill be included in separate containers or mixed in a single containerin desired ratios such as the ratios provided above. As previouslyindicated these ratios generally provide for relatively large amounts ofthe first protein such as the peptide of SEQ ID NO:8 and relativelysmall amounts of the second protein such as rhBMP-2.

Bone Morphogenic Protein

The second protein used in formulations of the present invention can bea naturally occurring protein or a recombinantly produced version ofsuch a naturally occurring protein. An example of such a protein is bonemorphogenic protein-2 (BMP-2) which in its recombinant version isreferred to as recombinant human bone morphogenic protein-2 (rhBMP-2).Information relating to bone morphogenic protein is contained within thefollowing publications which are incorporated herein by reference.BioDrugs. 2002; 16(5): 376-7. Journal of Orthopaedics 2005;2(4)e3; U.S.Pat. Nos. 5,108,922; 5,187,07; 5,318,898; 5,459,047; 5,618,924;5,631,142.

Bone morphogenic protein along with a first protein of the invention maybe combined with a suitable pharmaceutically acceptable carrier such asthe type I bovine absorbable collagen sponge (ACS).

A therapeutic formulation of the present invention is applied to thesites of bone and/or cartilage damage (e.g., bone fractures,osteotomies, etc.), thus providing localized delivery of the therapeuticprotein composition of the invention. For example, a therapeutic proteincomposition can be applied either by injection in a suitable carrier(e.g., an oily solvent such as arachis oil or an injectible bonecements) to the site of interest or, in cases of open surgery, by localapplication thereto of such compounds in a suitable carrier such asbone-wax, demineralized bone powder, polymeric bone cements, bonesealants, etc. Alternatively, local application can be achieved byapplying a solution or dispersion of the therapeutic protein compositionin a suitable carrier onto the surface of, or incorporating it intosolid or semi-solid implants conventionally used in orthopedic surgery,such as prostheses, dacron-mesh, Gore-tex™, gel-foam and kiel bone,and/or a collagen sponge/matrix. For example, absorbable collagensponges reconstituted from bovine tendon and collagen based matricesderived from demineralized/guanidine-extracted bovine bone are twodelivery materials currently being used for delivery of BMP to sites ofinterest (see e.g., Infuse™, Medtronic, Inc.; InductOs™, Wyeth/AstellasBV).

In certain embodiments, a therapeutic formulation of the inventionincludes a matrix capable of delivering a therapeutic proteincomposition such as SEQ ID NO:8 with rhBMP-2 to the site of bone and/orcartilage damage while providing a structure for the developing bone andcartilage. Such matrices may be formed of any convenient materialincluding those materials presently in use for other implanted medicalapplications. Such formulations may desirably be applied to a spongewhich is implanted, encapsulated or injected in a viscous form fordelivery to the site of bone, cartilage or tissue damage. The choice ofmatrix material can be based on several factors, includingbiocompatibility, biodegradability, mechanical properties, cosmeticappearance and interface properties. The particular application of aspecific therapeutic formulation will impact its design andimplementation. Potential matrices for the compositions may bebiodegradable and chemically defined calcium sulfate,tricalciumphosphate, hydroxyapatite, polylactic acid and polyanhydrides.Other potential materials are biodegradable and biologically welldefined, such as bone or dermal collagen. Further matrices are comprisedof pure proteins or extracellular matrix components. Other potentialmatrices are nonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalciumphosphate. The bioceramics may be altered in composition, suchas in calcium-aluminate-phosphate and processing to alter pore size,particle size, particle shape, and biodegradability.

The dosage regimen for therapeutic formulations of the present inventionsuch as SEQ ID NO:8 with rhBMP-2 will be determined by a number offactors which can affect the action of the therapeutic proteincomposition of a formulation of the invention, including amount of boneweight desired to be formed, the site of bone damage, the condition ofthe damaged bone, the size of a wound, type of damaged tissue, thepatient's age, sex, and diet, the severity of any infection, time ofadministration and other clinical factors. The dosage may vary with thetype of vehicle, carrier, or matrix employed as well as the identity ofthe therapeutic protein or proteins in the formulation. Efficacy oftreatment can be monitored in the clinic by periodic assessment of bonegrowth and/or repair, e.g. using x-rays.

In certain embodiments, the total dose in weight of the therapeuticprotein composition of a formulation of the invention such as SEQ IDNO:8 with rhBMP-2 can be from microgram (fig) to milligram (mg)quantities (e.g., from 1 μg to 100 mg, including from 2 μg to 50 mg,such as from 10 μg to 25 mg, 100 μg to 10 mg, etc.). As indicated above,the total dose can be determined by a number of factors. In certainembodiments, the therapeutic protein composition will be present in theformulation at a concentration (i.e., dosage in weight of therapeuticprotein composition/weight of total formulation) that ranges from 0.1mg/ml to 5 mg/ml, including 0.5 mg/ml to 4 mg/ml, such as 1 mg/ml to 3mg/ml, e.g., 2 mg/ml.

Therapeutic protein compositions in formulations of the invention maycontain two or more proteins. In one embodiment the formulation includesa first protein which is a protein which is encompassed by the generalsequence of SEQ ID NO: 1, in combination with the second protein whichis chosen from molecules belonging to the transforming growth factor(TGF) and bone morphogenetic protein (BMP) family as well the epidermalgrowth factor (EGF), epithelial cell growth factor (ECGF), fibroblastgrowth factor (FGF), platelet derived growth factor (PDGF), insulin-likegrowth factor (IGF), and insulin-like growth factor binding protein(IGFBP) families. In certain of these embodiments, the ratio of thesecond protein to the first protein in therapeutic formulations of theinvention range from 1:1 or more to 1:5,000 or more, including from 1:1or more to 1:1,000 or more, such as from 1:2 or more to 1:500 or more.

Those skilled in the art will recognize that substantial additionalinformation relating to BMP which is also referred to herein as BMP-2and rhBMP-2 can be found within the literature including numerous issuedU.S. Patents. Further, specific formulations including other proteinswhich promote hard tissue formation growth can be found in theliterature in connection with the present invention a first proteinwhich is encompassed by SEQ ID NO: 1 is combined with any of theseproteins which promote hard tissue formation and growth in order toenhance the therapeutic effect while using the smaller amounts of theprotein.

Manufacture for a Method

The formulation and/or kit may be manufactured for carrying out a methodcomprising:

-   -   administering to a patient a first peptide comprising 10 to 50        amino acid residues having an amino acid sequence of        RGDBD(X)nSGZG (SEQ ID NO: 1), wherein B, X, and Z can be any        amino acid residue and n is any integer between 1 and 10; and    -   further administering to the patient a second protein chosen        from the group comprising a transforming growth factor (TGF), a        bone morphogenetic protein (BMP), epidermal growth factor (EGF),        epithelial cell growth factor (ECGF), fibroblast growth factor        (FGF), platelet derived growth factor (PDGF), insulin-like        growth factor (IGF), and an insulin-like growth factor binding        protein (IGFBP).

In the method the patient may be a human and the method may be carriedout in order to enhance hard tissue formation activity. The method maybe carried out to extend retention time of a phosphorylated Smadmolecule. The method may be carried out in order to reduce time requiredto dephosphoiylate Smad molecules. The method as claimed in claim 14,wherein the method is carried out in order to reduce time required todegrade phosphorylated Smad molecules in hard tissue forming cells.

The cells treated may be tissue forming cells chosen from the group of:(a) cells of a lineage of osteoblast wherein the hard tissue is in theform of bone tissue; (b) cells of a lineage of ameloblast wherein thehard tissue is in the form of enamel; (c) cells of a lineage ofodontoblast wherein the hard tissue is in the form of dentin; and (d)cells of a lineage of cementoblast wherein the hard tissue is in theform of cementum.

The first peptide and the second peptide may be administered byinjection. The first peptide may be a peptide of SEQ ID NO:8 and thesecond peptide is a recombinant human bone morphogenic protein-2(rhBMP-2). The first protein and the second protein may be administeredat the same time. The first protein and the second protein may beadministered on an absorbable collagen sponge (ACS).

The formulation and/or kit may be manufactured for treating a diseasecharacterized by a hard tissue defect comprising:

(a) administering a peptide comprising about 10 to 50 amino acidscontaining an amino acid sequence of RGDBD(X)nSGZG (SEQ ID NO: 1), whereB. X, and Z can be any amino acid residue and n is any integer between 1and 10; and

(b) administering a molecule that promotes growth of a hard tissue, anda carrier.

The method may be carried out wherein (a) and (b) are administeredsimultaneously. The method may be carried out wherein (a) and (b) areadministered sequentially with the peptide being administered prior tothe molecule that promotes growth of a hard tissue. The method may becarried out wherein (b) and (a) are administered sequentially with themolecule that promotes growth of hard tissue being administered prior tothe peptide. The method may be carried out wherein administering (a) and(b) upregulates and/or activates cyclooxygenase-2 in the treated hardtissue. The method may be carried out wherein prostaglandin E₂production is increased by the treated hard tissue. The method may becarried out wherein degradation time of phosphorylated Smad molecules isextended in treated hard tissue cells.

A kit and/or formulation is manufactured for carrying out a method ofenhancing a hard tissue forming cell comprising extending the retentiontime of phosphorylated Smad 1, 5, and 8, wherein enhancing promotesproliferation, differentiation, maturation, or mineralization.

A kit and/or formulation is manufactured for carrying out a method oftreating a disease characterized by a hard tissue defect comprising:

administering one or more pharmaceutically active molecules that arecapable of extending phosphorylated Smad molecules retention time inhard tissue forming cells.

A kit and/or formulation is manufactured for carrying out a method ofregenerating cartilage comprising:

administering to a patient a pharmaceutically active amount of acomposition comprising polypeptides having the sequence of SEQ ID No. 1as defined here.

A kit and/or formulation is manufactured for carrying out a method oftreatment, comprising:

administering to a patient a therapeutically effective amount of aformulation comprising a pharmaceutically acceptable carrier and apeptide chosen from a peptide of any of SEQ ID NO:1-SEQ ID NO:8 asdefined herein.

The method may further comprise repeatedly administering the formulationto the patient periodically over a period of time in a manner so as topromote growth of hard tissue.

The method may further comprise administering a second drug whichinitials hard tissue formation.

The method may be carried out wherein the second drug is a recombinantlyproduced bone morphogenetic protein (BMP).

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Increase of Alkaline Phosphatase (ALP) Activity in PrimaryOsteoblasts Under Suboptimal Concentration of BMP-2 with the Addition ofthe Peptide of SEQ ID No. 8 to the Culture

Materials and Methods: Primary osteoblasts collected from fetal mousecalvaria were cultured under 0 or 25 ng/mL BMP-2. The peptide of SEQ IDNo. 8 was added to this culture system at 0, 0.2, 0.4, 1.0, 2.0, and 4.0μM. The levels of ALP were evaluated on days 0, 3, and 7.

Results: As shown in FIG. 1, ALP activity in osteoblastic cells wassignificantly increased in the groups treated with BMP-2 and the peptideof SEQ ID No. 8 in a dose dependent manner. In the 2 μM of the peptideof SEQ ID No. 8 treatment group, ALP activity was increased about 3times as compared with that in the control group and 2 times as comparedwith that in the BMP-2 treated cells without addition of the peptide.

Discussion: These results demonstrated that the peptide of SEQ ID No. 8significantly increased the ability of BMP-2 to positively influence thedifferentiation of pre-osteoblastic cells into osteoblasts.

Example 2 Increase in COX2 Levels in Primary hMSC with Addition ofDifferent Doses of the Peptide of SEQ ID No. 8

Materials and Methods: The effect of the peptide of SEQ ID No. 8treatment on gene expression was assessed in the Clonetics hMSC cellsusing gene expression microarray analysis as previously described(Locklin et al., 2001). Briefly, cells were plated in five T-150 ventedflasks at a density of 1.0×106 cells per flask in growth medium andcultured at 37° C. for 48 hours. Flasks containing either vehicle aloneor the peptide of SEQ ID No. 8 (1000 ng/ml) were cultured for 24 hr orfor 48 h in standard differentiation medium. At each time point, thevehicle and treatment flasks were trypsinized, cells removed and totalRNA was extracted for analysis via the Qiagen Rneasy Kit (Qiagen). 8 μgof total RNA were amplified and biotin labeled. The mixture washybridized to a U95A human gene expression microarray (Affymetrix)containing probes for 12,600 human genes. The chips were then washed,stained with phycoerythrin-streptavidin and read with an Affymetrixscanner. Hybridization intensity values to mRNA frequency werecalculated in molecules per million. Analysis of intensity values wasconducted using Spotfire Decision Site (Somerville, Mass.) software.

Results: Only mRNAs that were changed by the treatment with the peptideof SEQ ID NO. 8 by 2-fold or more were considered. The only mRNA thatwas changed by this extent at both time intervals was COX-2: it wasincreased at 24 h by 3.6-fold and at 48 h by 2.2-fold.

Discussion: These data demonstrated that the peptide of SEQ ID No. 8increased (by ˜3-fold) the mRNA for cyclooxgenase-2 (COX-2), aninducible enzyme required for prostaglandin synthesis. Thus the peptideof SEQ ID NO. 8 has the potential to induce the synthesis of PGE2 whichis known to have positive effects on bone formation.

Example 3 Potentiation of BMP-2 Stimulated PGE₂ Production by MC3T3Cells by the Peptide of SEQ ID No.8

Methods: MC3T3-E1 subclone 4 cells are neonatal mouse derived calvarialpreosteoblasts with fibroblast morphology. This subclone exhibits highlevels of osteoblast differentiation when grown in the presence ofascorbic acid and inorganic phosphate. For this experiment the MC3T3cells were plated 105 cells per well in 24 well plates in growth medium(alpha MEM+1 mM sodium pyruvate+10% fetal bovine serum (FBS)). After 24hours of culture the media was removed and replaced with assay media(alphaMEM+1 mM sodium pyruvate+2% FBS) containing the test substances.Each test group utilized 4 wells. After incubation for a further 24hours the media was removed and the levels of PGE2 were determined usingan ELISA (R&D Systems).

Results: The peptide of SEQ ID No. 8 increases the BMP-2 induced PGE2production as exhibited by FIG. 2.

Example 4 Retention of BMP-Activated Phosphorylated Smad 1, 5, and 8 inPrimary Mouse Osteoblasts When the Peptide of SEQ ID No. 8 Was Added tothe Cell Culture

Materials and Methods: Primary osteoblasts collected from fetal mousecalvaria were cultured with 0 or 25 ng/mL of BMP-2 with the addition of0 or 2 μM of the peptide of SEQ ID No. 8 to the culture system.Phosphorylation of Smad1/5/8 was evaluated by Western blotting at 0,0.5, 1, 2, 12, 24, 48 and 72 hours.

Results: Under these conditions BMP-2 induced phosphorylation ofSmad1/5/8, which was detectable from 0.5 to 12 hrs. The addition of 2 μMof the peptide of SEQ ID No. 8 to BMP-2 induced a prolongedphosphorylation of Smad1/5/8 which was detectable from 0.5 to 48 hrs(FIG. 3).

Discussion: These data indicated that the ability of the peptide of SEQID NO. 8 to potentate the activity of BMP-2 may be due at least in parton the ability of the peptide to significantly prolong the half-life ofthe phosphorylated Smad1/5/8 proteins, which are integral part of thesignal transduction pathway of BMP-2 and other bone anabolic agents.

Example 5 Increase in the Production of PGE₂ in Mouse Calvaria OrganCulture Assay with Suboptimal Concentration of BMP-2 with Addition ofdifferent Concentration of the Peptide of SEQ ID No. 8

Methods: Calvarial organ cultures have been used for many years to studythe regulation of bone formation and bone resorption. An advantage ofthis method compared to in vivo studies is the absence of confoundingeffects (such as hormonal and mechanical influences) meaning a moredirect effect of the test substances on bone tissue can be determined.Also compared to isolated cell systems, interrelationships between thedifferent cell types in the bone and between these cells and the bonematrix are preserved.

Briefly, hemi-calvariae were isolated from 4 day old Swiss Webster miceand incubated in media on steel mesh grids. The peptide of SEQ ID NO. 8(1, 10, 100 μg/mL) was added daily to the culture for seven days and aBMP-2 (40 ng/mL) control group was treated at days 1 and 4. Samples ofthe conditioned media were collected daily for analysis of PGE2concentration (R&D Systems ELISA kit). At the end of the culture thebones were analyzed for new bone formation and number of osteoblastsusing histology.

Results: The groups treated with the peptide of SEQ ID NO. 8 hadsignificant increases in PGE2 production (FIG. 4) and parameters of newbone formation, osteoblast surface and bone cell density (FIG. 5).

Discussion: These results indicate that the peptide of SEQ ID No. 8could enhance the ability of endogenous bone factors present in thecalvaria bone organ (including BMP2) to increase parameters of boneformation through an increased PGE2 production mechanism.

Example 6 Induction of the Bone Anabolic Activity of BMP-2 in MouseDorsal Muscle Using an Implantable Polymer Containing BMP-2 and thePeptide of SEQ ID No. 8

Materials and Methods: Polymer pellets containing both BMP-2 and thepeptide of SEQ ID No. 8 in the following amounts (see table below) wereimplanted in the dorsal subfascia of 4 week old ICR mice. The mice weresacrificed 3 weeks later and the newly formed bones were isolated forevaluation using soft X-ray radiograph and bone mineral densitydetermination.

N = 10 AC-100 (μg) 0 50 250 1250 BMP-2 (μg) 5 5 5 5 BMP-2 (μg) 3 3 3 3Polymer (mg) 30 30 30 30

Results: As exhibited in FIG. 6, the amount of bone formation wasevaluated by the size and mineral density of ectopic ossicles at threeweeks after implantation of 30 mg polymer containing either 5 μg or 3 μgof BMP-2 with different doses of the peptide of SEQ ID No. 8 (0, 50,250, or 1250 μg) into the dorsal muscle of mice (ICR). The efficacy ofthe peptide was unclear when 5 μg of BMP-2 was used. However, thepeptide showed significant enhancement of the BMP-2 effects when 3 μg ofBMP-2 was used.

Discussion: These data demonstrated that the peptide of SEQ ID No. 8 cansignificantly induce the bone anabolic effect of BMP-2 in vivo,especially when suboptimal doses of BMP-2 are used

Sequence Listing

RGDBD(X)nSGZG [B, X, and Z can be any amino acid, (SEQ ID NO.1) n= 1~10]] RGDND(X)nSGZG [X and Z can be any amino acid, n = (SEQ ID NO.2)1~10] RGDND(X)nSGDG [X can be any amino acid, n = 1~10] (SEQ ID NO.3)RGDNDJJPFSGDG [J can be any amino acid] (SEQ ID NO:4) RGDNDISPFSGDG (SEQID NO.5) RGDNDMSPFSGDG (SEQ ID NO.6) RGDNDVPPFSGDG (SEQ ID NO.7)TDLQERGDNDISPFSGDGQPFKD (SEQ ID NO.8)

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1. A method of treatment, comprising: administering to a patient a firstpeptide comprising 10 to 50 amino acid residues having an amino acidsequence of RGDBD(X)_(n)SGZG (SEQ ID NO:1), wherein B, X, and Z can beany amino acid residue and n is any integer between 1 and 10; andfurther administering to the patient a second protein chosen from thegroup comprising a transforming growth factor (TGF), a bonemorphogenetic protein (BMP), epidermal growth factor (EGF), epithelialcell growth factor (ECGF), fibroblast growth factor (FGF), plateletderived growth factor (PDGF), insulin-like growth factor (IGF), and aninsulin-like growth factor binding protein (IGFBP).
 2. The method asclaimed in claim l, wherein the patient is a human and the method iscarried out in order to enhance hard tissue formation activity.
 3. Themethod as claimed in claim 1, wherein the method is carried out toextend retention time of a phosphorylated Smad molecule.
 4. The methodas claimed in claim 1, wherein the method is carried out in order toreduce time required to dephosphoiylate Smad molecules.
 5. The method asclaimed in claim 1, wherein the method is carried out in order to reducetime required to degrade phosphorylated Smad molecules in hard tissueforming cells.
 6. The method of claim 5 wherein the tissue forming cellsare cells chosen from the group of: (a) cells of a lineage of osteoblastwherein the hard tissue is in the form of bone tissue; (b) cells of alineage of ameloblast wherein the hard tissue is in the form of enamel;(c) cells of a lineage of odontoblast wherein the hard tissue is in theform of dentin; and (d) cells of a lineage of cementoblast wherein thehard tissue is in the form of cementum.
 7. The method of claim 1,wherein the first peptide and the second peptide are administered byinjection.
 8. The method of claim 1, wherein the first peptide is apeptide of SEQ ID NO:8 and the second peptide is a recombinant humanbone morphogenic protein-2 (rhBMP-2).
 9. The method of claim 8, whereinthe first protein and the second protein are administered at the sametime.
 10. The method of claim 9, wherein the first protein and thesecond protein are administered on an absorbable collagen sponge (ACS).11. A method of treating a disease characterized by a hard tissue defectcomprising: (a) administering a peptide comprising about 10 to 50 aminoacids containing an amino acid sequence of RGDBD(X)_(n)SGZG (SEQ ID NO:1), where B, X, and Z can be any amino acid residue and n is any integerbetween 1 and 10; and (b) administering a molecule that promotes growthof a hard tissue, and a carrier.
 12. The method of claim 11 wherein (a)and (b) are administered simultaneously.
 13. The method of claim 11wherein (a) and (b) are administered sequentially with the peptide beingadministered prior to the molecule that promotes growth of a hardtissue.
 14. The method of claim 11 wherein (b) and (a) are administeredsequentially with the molecule that promotes growth of hard tissue beingadministered prior to the peptide.
 15. The method of claim 11 whereinadministering (a) and (b) upregulates and/or activates cyclooxygenase-2in the treated hard tissue.
 16. The method of claim 15 whereinprostaglandin E₂ production is increased by the treated hard tissue. 17.The method of claim 16 wherein degradation time of phosphorylated Smadmolecules is extended in treated hard tissue cells.
 18. A method ofenhancing a hard tissue forming cell comprising extending the retentiontime of phosphorylated Smad 1, 5, and 8, wherein enhancing promotesproliferation, differentiation, maturation, or mineralization byadministering a formulation comprising: a pharmaceutically acceptablecarrier; a first peptide comprising 10-50 amino acid residues having anamino acid sequence of RGDBD(X)_(n)SGZG (SEQ ID NO: 1), wherein B, X,and Z can be any amino acid residue and n is any integer between 1 and10; and a second peptide chosen from the group comprising a transforminggrowth factor (TGF), a bone morphogenetic protein (BMP), epidermalgrowth factor (EGF), epithelial cell growth factor (ECGF), fibroblastgrowth factor (FGF), platelet derived growth factor (PDGF), insulin-likegrowth factor (IGF), and an insulin-like growth factor binding protein(IGFBP).
 19. A method of treating a disease characterized by a hardtissue defect comprising: administering one or more pharmaceuticallyactive molecules that are capable of extending phosphorylated Smadmolecules retention time in hard tissue forming cells by administering aformulation comprising: a pharmaceutically acceptable carrier; a firstpeptide comprising 10-50 amino acid residues having an amino acidsequence of RGDBD(X)_(n)SGZG (SEQ ID NO: 1), wherein B, X, and Z can beany amino acid residue and n is any integer between 1 and 10; and asecond peptide chosen from the group comprising a transforming growthfactor (TGF), a bone morphogenetic protein (BMP). epidermal growthfactor (EGF), epithelial cell growth factor (ECGF), fibroblast growthfactor (FGF), platelet derived growth factor (PDGF), insulin-like growthfactor (IGF), and an insulin-like growth factor binding protein (IGFBP).20. A method of regenerating cartilage comprising: administering to apatient a pharmaceutically active amount of a composition comprisingpeptide comprising 10-50 amino acid residues having an amino acidsequence of RGDBD(X)_(n) 948 SGZG (SEQ ID NO:1), wherein B, X, and Z canbe any amino acid residue and n is any integer between 1 and
 10. 21. Amethod of treatment, comprising: administering to a patient atherapeutically effective amount of a formulation comprising apharmaceutically acceptable carrier and a peptide chosen from a peptideof any of SEQ ID NO:1-SEQ ID NO:8 as defined herein.
 22. The method asclaimed in claim 2 1, further comprising: repeatedly administering theformulation to the patient periodically over a period of time in amanner so as to promote growth of hard tissue.
 23. The method as claimedin claim 22, further comprising: administering a second drug whichinitials hard tissue formation.
 24. The method as claimed in claim 23wherein the second drug is a recombinantly produced bone morphogeneticprotein (BMP).